The plasma membrane of most eukaryotic cells is rich in cholesterol whereas internal membranes have very little sterol. The objective of the proposed research is the elucidation of the mechanisms governing this non-uniform distribution of cholesterol. A novel method for the measurement of cholesterol transfer between various intracellular pools and the plasma membrane will be applied in a study of fibroblasts. The method is based on the use of the enzyme cholesterol oxidase which catalyzes the oxidation of plasma membrane cholesterol to cholestenone in intact cells, while leaving cholesterol in internal pools untouched. Thus cholesterol in the plasma membrane can be separated and distinguished from intracellular cholesterol. The distribution and transfer of radiocholesterol between pools will be examined in cells labeled internally by cholesterol biosynthesis from [3H]acetate and labeled at the cell surface with exogenous [14C]cholesterol. The rate of transfer to the plasma membrane of radiocholesterol introduced into lysosomes by the receptor-mediated endocytosis of low density lipoprotein labeled with [3H]cholesteryl ester also will be measured by this technique. Equilibrium density gradient centrifugation of double-labeled cholesterol oxidase-treated cells will be used to examine the transfer of label between various intracellular pools. The power of this technique lies in the fact that it provides the means to eliminate the massive background contribution of plasma membrane throughout the gradient by selectively converting its cholesterol to cholestenone. Using this methodology, the following questions will be addressed: 1) What is the mechanism involved in the transport of newly synthesized cholesterol to the plasma membrane? 2) It is the case that cholesterol introduced exogenously into the plasma membrane does not equilibrate with intracellular pools? 3) Is there a common pathway of transfer to the plasma membrane of biosynthetic cholesterol and cholesterol which enters the cell by receptor-mediated endocytosis of low density lipoprotein? The extraordinary role of cholesterol in human cardiovascular disease warrants a full elucidation of these issues.